This project concerns mechanisms of CNS demyelination in human diseases and continues to focus on the molecular characterization of the human polyomavirus JC virus (JCV), the etiologic agent of progressive multifocal leukoencephalopathy (PML). PML is an infection of oligodendrocytes in the CNS, causing a demyelinating disease which is fatal in about 5% of AIDS patients, usually within 3-6 months. Work this year has continued to emphasize the detection and characterization of JCV by polymerase chain reaction (PCR) amplification from PML tissues, CSF, and from the urine of normal controls and multiple sclerosis (MS) patients. Notable advances this year have included the following: 1) the characterization of JCV and BKV in African urines obtained from AIDS patients in Shirati, Tanzania, including the identification of East African JCV as a new type of the virus (Type 3). The entire genome of several African strains has been amplified by PCR as six separate fragments, and the genome of this new type will be sequenced; (2) A group of urines from 105 control individuals including 5 African-Americans was characterized for the type of JCV present. These were typed as follows: 29 Type 1 strains (1A or 1B), eight Type 2 strains, no Type 3 strains, seven Type 4 strains, and one Type 5 strain. Two individuals were doubly infected with different types or subtypes of JCV. Two possible new types of JCV were identified in these patients. Type 4 consists of an insert of about 150bp from Type 3 into Type 1 sequence. Type 5, with a single example discovered so far, may be derived from a sequence ancestral to Types 2 and 3 to which it is most closely related. (3) Chronic progressive MS patients showed numbers of JCV positive urines comparable to controls, and excretion of the virus was not enhanced by immunosuppressive treatment with cyclosporine. No examples of Type 2 were identified in a group of 37 patients, but this difference in type distribution compared to controls was not statistically significant. Larger numbers of patients, including those with relapsing/remitting disease, will be studied, and the influence of clinical relapse on virus excretion will be examined.( 4) A technical advance developed in our Section has allowed cloning the entire JCV genome (5100 bp) from brain, CSF, and urine. This will greatly facilitate cloning and sequencing of variant JCV strains, including those derived from MS patients.